In modern agriculture, being able to detect a specific nucleotide sequence of a so-called nucleic acid of interest or target sequence, is becoming more and more important. This capability allows e.g. to rapidly detect specific nucleic acid sequences associated with the presence of a particular characteristic or trait in a plant, thereby allowing to develop particular plants with particular combinations of characteristics in a more direct and more efficient manner. Such capability also allows to detect particular variant alleles in plants.
With the development of transgenic plants, a need has arisen to be able to detect the presence of biological material comprising particular transgenic events, e.g. in the field, in the variety development of plants, or in the commercial chain (grain production, grain transport, grain storage etc.), preferably at higher speed, lower cost and with greater versatility. Moreover, different transgenic events may comprise similar or even identical nucleic acids, and often it is desirable to be able to distinguish between such different transgenic events, comprising similar or identical nucleic acids, requiring the application of event specific detection methods and tools.
Additionally, more and more plant diseases can be rapidly and unambiguously diagnosed via detection of specific nucleotide sequences, associated with the pathogens (fungal, viral, bacterial, nematode or other plant pests) causing the disease.
The applicability of detection of specific nucleotide sequence in biological material is of course not limited to agricultural applications, but also extends into other fields, including the medical field, forensics, genetic counseling etc.
Various detection methods are based upon amplification of a target nucleic acid and/or DNA having a specific nucleotide sequence, the oldest process being the polymerase chain reaction.
Of particular interest are the isothermal DNA amplification methods, including the so-called LAMP or Loop-mediated Isothermal Amplification as described in e.g. U.S. Pat. No. 641,027 (Eiken) The method is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions on the target gene and the reaction process proceeds at a constant temperature using strand displacement reaction. Amplification and detection of target nucleic acid of interest can be completed in a single step, by incubating the mixture of the biological sample or a nucleic acid extract thereof, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature (about 65° C.). It provides high amplification efficiency, with DNA being amplified 109-1010 times in 15-60 minutes. Because of its high specificity, the presence of amplified product can indicate the presence of target gene (http://loopamp.eiken.co.jp/e/lamp/index.html).
Other isothermal DNA amplification methods include the so-called Nicking Enzyme Amplification Reaction (NEAR) (Envirologix). NEAR uses a nicking enzyme and strand-displacing polymerase to generate small pieces of DNA that feed a DNA extension reaction; alternating cycles of nicking and extension lead to exponential amplification. The method is described in e.g. US 2009/0017453.
Yet another isothermal nucleic acid and/or DNA amplification method is the so-called Recombinase Polymerase Amplification (RPA) (TwistDx). The RPA method uses recombinases, which are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA. Through this method, DNA synthesis by a DNA polymerase is directed to defined points in a sample DNA. If the target sequence is present, a DNA amplification reaction is initiated. Recombinase polymerase amplification is described e.g. in U.S. Pat. No. 7,270,981.
Other isothermal amplification methods are described in Gill and Ghaemi, 2008, Nucleosides Nucleotides Nucleic Acids, 27(3) 224-243.
An important step in all nucleic acid and/or DNA amplification methods is the preparation of the template nucleic acids from the biological material. For automatic processing of the amplification reaction, convenient and efficient nucleic acid extraction from the biological samples, yielding sufficient template nucleic acids, and preferably in a solution without turbidity, is preferred. Preferably, the extraction step should only have minimal maceration, or even avoid mechanical maceration of the biological material as this may introduce turbidity in the solution. Alkaline extraction of template nucleic acid and/or DNA from biological samples may provide such a method using only minimal maceration of the biological sample.
Klimyuk (1993) (Plant Journal 3(3) 493-494) described alkali treatment for rapid preparation of plant material for reliable PCR analysis.
Chomczynski and Rymaszewski (2006) described and alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. (BioTechniques, 40, 454-457).
A drawback of the alkaline extraction methods, is that the resulting extract needs to be neutralized or diluted sufficiently, prior to adding the extract or an aliquot thereof to the amplification reaction mixture. Such a dilution or neutralization step results in additional handling of the sample, increasing the risk of contamination of the sample with unwanted nucleic acids and/or DNA. The additional step further complicates automated processing of the amplification reaction (see also Lee et al., 2009, J. Agric. Food Chem. 2009, 57, 9400-9402).
Furthermore, there is a need for automated processing of sampling, amplification and detection with a minimum of process steps. Such kits and/or devices for automated processing could be used in environment outside of a laboratory, particularly if the chemistry and processing is robust.
Current detection methods to be used outside of a laboratory environment are protein based detection tools, such as Lateral Flow Strips. Protein based detection tools fail to detect e.g. transgenic plants with silenced genes causing a trait, temporal or special expression of proteins, or cannot distinguish between different plants or transgenic events expressing the same or similar proteins.
The current invention provides a solution to these problems as described hereinafter in the summary, detailed embodiments, examples, drawings and claims.